1. Field of the Invention
The present invention relates to an improved method for operating a mass spectrometer and, more specifically, it relates to controlling a generally constant ratio of the amplitude of the trapping voltage to the amplitude of the excitation voltage of a quadrupole ion trap and, more specifically, it relates to a method for calibrating an ion trap mass spectrometer in the resonance ejection mode and, most specifically, is particularly advantageous in calibrating a quadrupole ion trap mass spectrometer using a single datum point. The invention also relates to an improved mass spectrometer apparatus operating in the resonance ejection mode and, more specifically, it relates to controlling a generally constant ratio of the amplitude of the trapping voltage to the amplitude of the excitation voltage for mass analyzing ions.
2. Description of the Prior Art
The use of mass spectrometers in determining the identity and quantity of constituent materials in a gaseous, liquid or solid specimen has long been known. Mass spectrometers or mass filters typically use the ratio of the mass of an ion to its charge, m/z, for analyzing and separating ions. The ion mass m is typically expressed in atomic mass units or Daltons (Da) and the ion charge z is the charge on the ion in terms of the number of electron charges e.
It is known, in connection with mass spectrometer systems, to analyze a specimen under vacuum through conversion of the molecules into an ionic form, separating the ions according to their m/z ratio, and permitting the ions to bombard a detector. See, generally, U.S. Pat. Nos. 2,882,410; 3,073,951; 3,590,243; 3,955,084; 4,175,234; 4,298,795; 4,473,748; and 5,155,357. See, also U.S. Pat. Nos. 4,882,485; and 4,952,802.
It is known to use an ion trap mass spectrometer (ITMS) for mass analysis of large biological molecules and for tandem mass spectral measurements to provide structural and sequential information about peptides and other biopolymers. Known ionizers contain an ionizer inlet assembly wherein the specimen to be analyzed is received, a high vacuum chamber which cooperates with the ionizer inlet assembly, and an analyzer assembly which is disposed within the high vacuum chamber and is adapted to receive ions from the ionizer. Detector means are employed in making a determination as to the constituent components of the specimen employing the mass-to-charge ratio as a distinguishing characteristic. By one of a variety of known methods, such as electron impact (EI), the molecules of the gaseous specimen contained in the ionizer are converted into ions for subsequent analysis.
It is also known to use desorption methods for ionizing large molecules. Such methods include secondary ion mass spectrometry, fast-atom bombardment, electrospray ionization (ESI) in which ions are evaporated from solutions, laser desorption, and matrix-assisted laser desorption/ionization (MALDI). In the MALDI desorption method, biomolecules to be analyzed are recrystallized in a solid matrix of a low mass chromophore. Following absorption of the laser radiation by the matrix, ionization of the analyte molecules occurs as a result of desorption and subsequent charge exchange processes. See Doroshenko, V. M. et al., "High-Resolution Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biomolecules in a Quadrupole Ion Trap," Laser Ablation: Mechanisms and Applications--II, Second International Conference, pp. 513-18, American Institute of Physics (1993).
Known mass analyzers come in a variety of types, including magnetic field (B), combined electrical and magnetic field or double-focusing instruments (EB and BE), quadrupole electric field (Q), and time-of-flight (TOF) analyzers. In addition, two or more analyzers may be combined in a single instrument to produce tandem (MS/MS or MS/MS/MS, for example) or hybrid mass spectrometers such as, for example, triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and other hybrids (e.g., EBqQ). Such known tandem and hybrid instruments require the use of additional mass analyzers. For example, in a triple quadrupole, a first quadrupole is used as a mass filter to select ions of a given mass, a second quadrupole is used as a collision chamber for fragmenting the selected ions, and a third quadrupole is used for mass analyzing the fragmented ions.
Ion traps are capable of storing one or more kinds of ions for relatively long periods of time. In contrast to the tandem and hybrid instruments, the ion trap separates successive reaction steps in time rather than in space.
A known design of a quadrupole ion trap mass spectrometer consists of a central, hyperbolic cross-section, ring electrode located between two hyperbolic end-cap electrodes. In the known EI ionization method, ions are trapped and confined inside the ion trap cell by applying a radio frequency (RF) voltage on the ring electrode with the end-cap electrodes grounded. Ions of different m/z ratios are trapped simultaneously. It is known to determine the mass range of the trapped ions by an ion stability diagram, such as the one shown in FIG. 1, using the dimensionless Mathieu parameters (a.sub.z and q.sub.z) which depend upon the radius of the trap (r.sub.o), the direct current (DC) voltage (U) and RF voltage (V) amplitudes, and the RF frequency (F=.OMEGA./2 .pi.).
In the known mass selective instability operating mode, ions move along the q.sub.z axis (with U=q.sub.z =0 from the left to the right in FIG. 1) with increasing RF voltage V amplitude. Ions of increasingly higher mass arrive at the stability border in succession, exit the trap in the z (axial) direction, and are detected by a multiplier located behind one of the end-cap electrodes. In this mode, ions become unstable in the strong RF trapping field.
A known technique for extending the mass range of the quadrupole ion trap is the axial resonant ejection operating mode or resonance ejection mode. A bipolar, supplementary, low amplitude RF excitation voltage is applied to the end-cap electrodes. Ions are excited and ejected from the quadrupole ion trap with the use of a supplementary, weak dipole resonant electric field. In this mode, ions are selected for ejection along the q.sub.z axis lying within the stability diagram of FIG. 1 by the applied supplementary RF field across the end-cap electrodes. A wide range of masses may be ejected by using an appropriate choice of the frequency of the excitation voltage. See, generally, U.S. Pat. No. Re. 34,000.
An equilibrium condition of the amplitude of ion oscillation occurs whenever the power gained by the ion oscillator from the excitation field is equal to the power lost in the collisions with a buffer gas. If absorption takes place at the wing of the absorption contour, then the amplitude A of the ion oscillatory motion is determined by Equation 1: ##EQU1## wherein: F.sub.s =zev.sub.s /2.sup.1/2 f.sub.o is excitation force
z is ion charge PA1 e is electron charge PA1 v.sub.s is excitation voltage amplitude PA1 r.sub.o is radius of the ring electrode PA1 m is ion mass PA1 .omega..sub.s =2.pi.f.sub.s is excitation voltage frequency PA1 f.sub.s is excitation voltage frequency PA1 .tau. is effective time between ion-neutral collisions describing damping of the ion oscillator PA1 a=d.omega./dt is secular frequency scan rate PA1 .omega. is secular frequency of ion oscillation PA1 t is time with t=0 corresponding to .omega.=.omega..sub.s
Equation 1 is valid whenever the secular frequency is scanned linearly (i.e., .DELTA..omega.=.omega..sub.s -.omega.=-at&gt;&gt;1/.tau.) or whenever the secular frequency scan rate is relatively low (i.e., a.sup.1/2 .tau.&lt;&lt;1).
Kaiser, R. E., Jr. et at., "Operation of a Quadrupole Ion Trap Mass Spectrometer to Achieve High Mass/Charge Ratios", International Journal of Mass Spectrometry and Ion Processes, 106 (1991) 79-115, discloses the possibility of using amplitude modulation by the excitation voltage for the extension of the mass range. Because the process of resonance excitation takes some time and the ion oscillator has a finite frequency range for excitation, the free oscillation frequency of ions at the time of ejection does not correspond to the excitation frequency. This results in an apparent mass shift with respect to the ideal situation in which the secular oscillation and excitation frequencies coincide at the ejection time. As shown in FIG. 21 of Kaiser, Jr. et at., as the amplitude of the axial modulation voltage is varied, the shift in mass becomes more pronounced. The dependence of the mass shift upon the excitation voltage amplitude is not completely linear. At lower masses, there is a larger absolute mass shift and, alternatively, at higher masses, there is a smaller mass shift. Above a certain threshold, the mass shift is approximately linear, but not proportional, with increasing axial modulation voltage.
When using axial modulation for mass range extension, a substantial mass shift, which is a function of the frequency and amplitude of the supplementary voltage, is observed. In order to achieve a linear calibration for a mass spectrum, the apparent mass shift of an ion must be independent of the chosen mass range (e.g., 0-70,000 Da). As shown in FIG. 22 of Kaiser, Jr. et al., a linear relationship is observed between the apparent mass shift and the mass of the ion at high mass scan rates and constant amplitude of the excitation voltage. By ramping the excitation voltage linearly with the RF trapping scan, a constant mass shift with respect to the ion mass can be achieved if relatively large amplitudes of the excitation voltage are used.
It has been known with prior art ion cyclotron resonance spectrometers to provide a frequency of a trapping RF voltage which is twice as high as the resonance frequency of the trapping oscillation of charged particles. See, generally, U.S. Pat. No. 4,818,864.
It has been known with prior art cycloidal mass spectrometers to use a single fixed collector and a ramped electric field in looking at only one mass-to-charge ratio at a time. See, generally, U.S. Pat. No. 5,304,799.
It has been known with prior art quadrupole mass filters to apply an excitation voltage having both a DC component (U) and an AC component (V) to four primary electrodes and to provide a DC voltage (-U'), which is directly proportional to the DC component (U), between a guard electrode and an intermediate electrode. See, generally, U.S. Pat. No. 3,617,736.
It has also been known with prior art quadrupole mass filters, which transmit particles having a selected mass-to-charge ratio, to provide a power supply which maintains a constant ratio of the amplitude of the DC potential applied to four elongate filter electrodes to the amplitude of the RF potential applied to such electrodes. See, generally, U.S. Pat. No. 5,354,988.
It has been known with prior art ion trap mass spectrometers to vary the amplitude, frequency or direct potential of the trapping RF voltage. See, generally, U.S. Pat. No. 5,028,777.
It has also been known with prior art ion trap mass spectrometers to set the amplitude of the excitation voltage proportional to the square root of the amplitude of a storage (trapping) RF voltage. See, generally, U.S. Pat. No. 5,298,746.
Known mass spectrometers operating in the resonance ejection mode attempt to achieve linear mass calibration over specific mass ranges but do not provide a mass calibration which is independent of the mass scan range. In a prior publication concerning known mass spectrometers, it has been suggested that the amplitude of the excitation voltage be scanned linearly, but not directly proportional, to the amplitude of the trapping RF voltage.
Known methods of calibration in the resonance ejection mode include the external and internal calibration methods. In the external method, calibration curves are generated using well known calibrant masses before the experiment in which the unknown substances are analyzed. See, for example, Kaiser, Jr. et at. In the internal method, the calibrant and analyte ions are recorded simultaneously in the same experiment. This method achieves a better mass assignment accuracy than the external method because all ions are in the same environmental conditions. See, for example, Williams, J. D. et at., "Improved Accuracy of Mass Measurement with a Quadrupole Ion-Trap Mass Spectrometer", Rapid Communications in Mass Spectrometry, 6 (1992) 524-27.
Because the known dependence of the mass shift upon the excitation voltage amplitude is not completely linear, linear modulation of the excitation voltage cannot compensate for the mass shift due to changing mass. Furthermore, because the calibration curve is not completely linear, both the external and internal calibration methods require plural calibration compounds which produce a series of calibrant peaks repeated by small intervals in order to provide good accuracy. The optimum value of the excitation voltage is usually determined by the requirements for sensitivity and/or mass resolution. Because the optimum excitation voltage usually increases with mass, a new calibration is typically necessary for every mass subregion.
For these reasons, there remains a very real and substantial need for an improved mass spectrometer and calibration method therefor. In particular, there is a very real and substantial need for an internal calibration method for the ESI and MALDI ionization methods, which are typically applied to biomolecules having widely disparate m/z peaks, where simultaneous generation of analyte and calibrant ions is known to be a difficult task.